Cyclosporin A inhibits PDGF-BB induced hyaluronan synthesis in orbital fibroblasts.

Orbital connective tissue changes are contributors to the pathogenesis in thyroid eye disease (TED). Activated fibroblasts respond to immune stimuli with proliferation and increased hyaluronan (HA) production. Cyclosporin A (CsA) was reported to be beneficial in the treatment of TED. PDGF isoforms are increased in orbital tissue of TED patients and enhance HA production. We aimed to study the effect of CsA on HA production and hyaluronan synthase (HAS1, 2 and 3) and hyaluronidase (HYAL1 and 2) mRNA expressions in orbital fibroblasts (OFs). Measurements were performed in the presence or absence of CsA (10 µM) in unstimulated or PDGF-BB (10 ng/ml) stimulated OFs. The HA production of TED OFs (n = 7) and NON-TED OFs (n = 6) were measured by ELISA. The levels of mRNA expressions were examined using RT-PCR. The proliferation rate and metabolic activity were measured by BrdU incorporation and MTT assays, respectively. Treatment with CsA resulted in an average 42% decrease in HA production of OFs (p < 0.0001). CsA decreased the expression levels of HAS2, HAS3 and HYAL2 (p = 0.005, p = 0.005 and p = 0.002, respectively.) PDGF-BB increased HA production (p < 0.001) and HAS2 expression (p = 0.004). CsA could reduce the PDGF-BB-stimulated HA production (p < 0.001) and HAS2 expression (p = 0.005) below the untreated level. In addition, CsA treatment caused a decrease in proliferation potential (p = 0.002) and metabolic activity (p < 0.0001). These findings point to the fact that CsA affects HA metabolism via HAS2, HAS3 and HYAL2 inhibition in OFs. In addition to its well characterized immunosuppressant properties, CsA's beneficial effect in TED may be related to its direct inhibitory effect on basal and growth factor stimulated HA production.

Hyaluronic Acid-Extraction Methods, Sources and Applications.

In this review, a compilation of articles in databases on the extraction methods and applications of hyaluronic acid (HA) was carried out. HA is a highly hydrated component of different tissues, including connective, epithelial, and neural. It is an anionic, linear glycosaminoglycan (GAG) primarily found in the native extracellular matrix (ECM) of soft connective tissues. Included in the review were studies on the extraction methods (chemical, enzymatical, combined) of HA, describing advantages and disadvantages as well as news methods of extraction. The applications of HA in food are addressed, including oral supplementation, biomaterials, medical research, and pharmaceutical and cosmetic industry applications. Subsequently, we included a section related to the structure and penetration routes of the skin, with emphasis on the benefits of systems for transdermal drug delivery nanocarriers as promoters of percutaneous absorption. Finally, the future trends on the applications of HA were included. This final section contains the effects before, during, and after the application of HA-based products.

Targeting BET Proteins Decreases Hyaluronidase-1 in Pancreatic Cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is characterized by the presence of dense stroma that is enriched in hyaluronan (HA), with increased HA levels associated with more aggressive disease. Increased levels of the HA-degrading enzymes hyaluronidases (HYALs) are also associated with tumor progression. In this study, we evaluate the regulation of HYALs in PDAC. METHODS: Using siRNA and small molecule inhibitors, we evaluated the regulation of HYALs using quantitative real-time PCR (qRT-PCR), Western blot analysis, and ELISA. The binding of BRD2 protein on the HYAL1 promoter was evaluated by chromatin immunoprecipitation (ChIP) assay. Proliferation was evaluated by WST-1 assay. Mice with xenograft tumors were treated with BET inhibitors. The expression of HYALs in tumors was analyzed by immunohistochemistry and by qRT-PCR. RESULTS: We show that HYAL1, HYAL2, and HYAL3 are expressed in PDAC tumors and in PDAC and pancreatic stellate cell lines. We demonstrate that inhibitors targeting bromodomain and extra-terminal domain (BET) proteins, which are readers of histone acetylation marks, primarily decrease HYAL1 expression. We show that the BET family protein BRD2 regulates HYAL1 expression by binding to its promoter region and that HYAL1 downregulation decreases proliferation and enhances apoptosis of PDAC and stellate cell lines. Notably, BET inhibitors decrease the levels of HYAL1 expression in vivo without affecting the levels of HYAL2 or HYAL3. CONCLUSIONS: Our results demonstrate the pro-tumorigenic role of HYAL1 and identify the role of BRD2 in the regulation of HYAL1 in PDAC. Overall, these data enhance our understanding of the role and regulation of HYAL1 and provide the rationale for targeting HYAL1 in PDAC.

Hyaluronan breakdown by snake venom hyaluronidases: From toxins delivery to immunopathology.

Snake venom enzymes have a broad range of molecular targets in plasma, tissues, and cells, among which hyaluronan (HA) is outstanding. HA is encountered in the extracellular matrix of diverse tissues and in the bloodstream, and its different chemical configurations dictate the diverse morphophysiological processes in which it participates. Hyaluronidases are highlighted among the enzymes involved in HA metabolism. This enzyme has been detected along the phylogenetic tree, suggesting that hyaluronidases exert multiple biological effects on different organisms. Hyaluronidases have been described in tissues, blood and snake venoms. Snake venom hyaluronidases (SVHYA) contribute to tissue destruction in envenomations and are called spreading factors since their action potentiates venom toxin delivery. Interestingly, SVHYA are clustered in Enzyme Class 3.2.1.35 together with mammalian hyaluronidases (HYAL). Both HYAL and SVHYA of Class 3.2.1.35 act upon HA, generating low molecular weight HA fragments (LMW-HA). LMW-HA generated by HYAL becomes a damage-associated molecular pattern that is recognized by Toll-like receptors 2 and 4, triggering cell signaling cascades culminating in innate and adaptive immune responses that are characterized by lipid mediator generation, interleukin production, chemokine upregulation, dendritic cell activation and T cell proliferation. In this review, aspects of the structures and functions of HA and hyaluronidases in both snake venoms and mammals are presented, and their activities are compared. In addition, the potential immunopathological consequences of HA degradation products generated after snakebite envenoming and their use as adjuvant to enhance venom toxin immunogenicity for antivenom production as well as envenomation prognostic biomarker are also discussed.

The role of hyaluronan in renal cell carcinoma.

Renal cell carcinoma (RCC) is associated with high mortality rates worldwide and survival among RCC patients has not improved significantly in the past few years. A better understanding of the pathogenesis of RCC can enable the development of more effective therapeutic strategies against RCC. Hyaluronan (HA) is a glycosaminoglycan located in the extracellular matrix (ECM) that has several roles in biology, medicine, and physiological processes, such as tissue homeostasis and angiogenesis. Dysregulated HA and its receptors play important roles in fundamental cellular and molecular biology processes such as cell signaling, immune modulation, tumor progression and angiogenesis. There is emerging evidence that alterations in the production of HA regulate RCC development, thereby acting as important biomarkers as well as specific therapeutic targets. Therefore, targeting HA or combining it with other therapies are promising therapeutic strategies. In this Review, we summarize the available data on the role of abnormal regulation of HA and speculate on its potential as a therapeutic target against RCC.

Hyaluronan breakdown by snake venom hyaluronidases: from toxins delivery to immunopathology

Snake venom enzymes have a broad range of molecular targets in plasma, tissues, and cells, among which hyaluronan (HA) is outstanding. HA is encountered in the extracellular matrix of diverse tissues and in the bloodstream, and its different chemical configurations dictate the diverse morphophysiological processes in which it participates. Hyaluronidases are highlighted among the enzymes involved in HA metabolism. This enzyme has been detected along the phylogenetic tree, suggesting that hyaluronidases exert multiple biological effects on different organisms. Hyaluronidases have been described in tissues, blood and snake venoms. Snake venom hyaluronidases (SVHYA) contribute to tissue destruction in envenomations and are called spreading factors since their action potentiates venom toxin delivery. Interestingly, SVHYA are clustered in Enzyme Class 3.2.1.35 together with mammalian hyaluronidases (HYAL). Both HYAL and SVHYA of Class 3.2.1.35 act upon HA, generating low molecular weight HA fragments (LMW-HA). LMW-HA generated by HYAL becomes a damage-associated molecular pattern that is recognized by Toll-like receptors 2 and 4, triggering cell signaling cascades culminating in innate and adaptive immune responses that are characterized by lipid mediator generation, interleukin production, chemokine upregulation, dendritic cell activation and T cell proliferation. In this review, aspects of the structures and functions of HA and hyaluronidases in both snake venoms and mammals are presented, and their activities are compared. In addition, the potential immunopathological consequences of HA degradation products generated after snakebite envenoming and their use as adjuvant to enhance venom toxin immunogenicity for antivenom production as well as envenomation prognostic biomarker are also discussed.

The hyaluronan-related genes HAS2, HYAL1-4, PH20 and HYALP1 are associated with prognosis, cell viability and spheroid formation capacity in ovarian cancer.

PURPOSE: Hyaluronan modulates tumour progression, including cell adhesion, cohesion, proliferation and invasion, and the cancer stem cell phenotype. In ovarian cancer, high levels of stromal hyaluronan are associated with poor prognosis. In this work, hyaluronan synthases (HAS1-3) and hyaluronidases (HYAL1-4, PH-20, HYALP1) were examined with regard to different levels of gene expression and its influence on ovarian cancer patients' survival. The impact of a siRNA depletion of HAS2 was investigated in vitro. METHODS: Using the Kaplan-Meier Plotter tool, we investigated the influence of hyaluronic synthases and hyaluronidases on the survival of a collective of 1435 ovarian cancer patients. Differences in gene expression between normal (n = 46) and cancerous (n = 744) ovarian tissue were examined using the TNMplot database. Following an evaluation of hyaluronan-related gene expression in the ATCC ovarian cancer panel, we studied SKOV3 and SW 626 ovarian cancer cells subjected to HAS2 siRNA or control siRNA treatment in terms of HAS1-3, HYAL2 and HYAL3 mRNA expression. We investigated the ability to form spheroids using the Hanging Drop method and the response to chemotherapy at different concentrations using the MTT Assay. By STRING analysis, interactions within the enzymes of the hyaluronic acid system and with binding partners were visualized. RESULTS: HAS1, HYAL1 and HYAL4 mRNA expression is significantly upregulated, whereas HAS2, HYAL2 and HYAL3 mRNA expression is significantly downregulated in ovarian cancer tissue compared to controls. HAS2 improves cell viability, the capability to form tumour spheroids and has a negative prognostic value regarding overall survival. Lower HAS2 expression and high expression of HYAL2 and HYAL3 favours the survival of ovarian cancer patients. HAS2 knockdown cells and control cells showed a moderate response to combinatorial in vitro chemotherapy with taxol and cisplatin. CONCLUSION: In conclusion, our study shows that the hyaluronic acid system has a relevant influence on the survival of ovarian cancer patients and could therefore be considered as a possible prognostic factor.

Hybridization between Crotalus aquilus and Crotalus polystictus Species: A Comparison of Their Venom Toxicity and Enzymatic Activities.

Hybridization is defined as the interbreeding of individuals from two populations distinguishable by one or more heritable characteristics. Snake hybridization represents an interesting opportunity to analyze variability and how genetics affect the venom components between parents and hybrids. Snake venoms exhibit a high degree of variability related to biological and biogeographical factors. The aim of this work is to analyze the protein patterns and enzymatic activity of some of the main hemotoxic enzymes in snake venoms, such as serine proteases (trypsin-like, chymotrypsin-like, and elastase-like), metalloproteases, hyaluronidases, and phospholipase A2. The lethal dose of 50 (LD50) of venom from the Crotalus aquilus (Cabf) and Crotalus polystictus (Cpbm) parents and their hybrids in captivity was determined, and phenetic analysis is also conducted, which showed a high similarity between the hybrids and C. polystictus. The protein banding patterns and enzymatic activity analyze by zymography resulted in a combination of proteins from the parental venoms in the hybrids, with variability among them. In some cases, the enzymatic activity is higher in the hybrids with a lower LD50 than in the parents, indicating higher toxicity. These data show the variability among snake venoms and suggest that hybridization is an important factor in changes in protein concentration, peptide variability, and enzymatic activity that affect toxicity and lethality.

Brown spider venom toxins: what are the functions of astacins, serine proteases, hyaluronidases, allergens, TCTP, serpins and knottins?

Accidents caused by the bites of brown spiders (Loxosceles) generate a clinical condition that often includes a threatening necrotic skin lesion near the bite site along with a remarkable inflammatory response. Systemic disorders such as hemolysis, thrombocytopenia, and acute renal failure may occur, but are much less frequent than the local damage. It is already known that phospholipases D, highly expressed toxins in Loxosceles venom, can induce most of these injuries. However, this spider venom has a great range of toxins that probably act synergistically to enhance toxicity. The other protein classes remain poorly explored due to the difficulty in obtaining sufficient amounts of them for a thorough investigation. They include astacins (metalloproteases), serine proteases, knottins, translationally controlled tumor proteins (TCTP), hyaluronidases, allergens and serpins. It has already been shown that some of them, according to their characteristics, may participate to some extent in the development of loxoscelism. In addition, all of these toxins present potential application in several areas. The present review article summarizes information regarding some functional aspects of the protein classes listed above, discusses the directions that could be taken to materialize a comprehensive investigation on each of these toxins as well as highlights the importance of exploring the full venom repertoire.

Analysis of High Molecular Mass Compounds from the Spider Pamphobeteus verdolaga Venom Gland. A Transcriptomic and MS ID Approach.

Nowadays, spider venom research focuses on the neurotoxic activity of small peptides. In this study, we investigated high-molecular-mass compounds that have either enzymatic activity or housekeeping functions present in either the venom gland or venom of Pamphobeteus verdolaga. We used proteomic and transcriptomic-assisted approaches to recognize the proteins sequences related to high-molecular-mass compounds present in either venom gland or venom. We report the amino acid sequences (partial or complete) of 45 high-molecular-mass compounds detected by transcriptomics showing similarity to other proteins with either enzymatic activity (i.e., phospholipases A2, kunitz-type, hyaluronidases, and sphingomyelinase D) or housekeeping functions involved in the signaling process, glucanotransferase function, and beta-N-acetylglucosaminidase activity. MS/MS analysis showed fragments exhibiting a resemblance similarity with different sequences detected by transcriptomics corresponding to sphingomyelinase D, hyaluronidase, lycotoxins, cysteine-rich secretory proteins, and kunitz-type serine protease inhibitors, among others. Additionally, we report a probably new protein sequence corresponding to the lycotoxin family detected by transcriptomics. The phylogeny analysis suggested that P. verdolaga includes a basal protein that underwent a duplication event that gave origin to the lycotoxin proteins reported for Lycosa sp. This approach allows proposing an evolutionary relationship of high-molecular-mass proteins among P. verdolaga and other spider species.

Key Matrix Remodeling Enzymes: Functions and Targeting in Cancer.

Tissue functionality and integrity demand continuous changes in distribution of major components in the extracellular matrices (ECMs) under normal conditions aiming tissue homeostasis. Major matrix degrading proteolytic enzymes are matrix metalloproteinases (MMPs), plasminogen activators, atypical proteases such as intracellular cathepsins and glycolytic enzymes including heparanase and hyaluronidases. Matrix proteases evoke epithelial-to-mesenchymal transition (EMT) and regulate ECM turnover under normal procedures as well as cancer cell phenotype, motility, invasion, autophagy, angiogenesis and exosome formation through vital signaling cascades. ECM remodeling is also achieved by glycolytic enzymes that are essential for cancer cell survival, proliferation and tumor progression. In this article, the types of major matrix remodeling enzymes, their effects in cancer initiation, propagation and progression as well as their pharmacological targeting and ongoing clinical trials are presented and critically discussed.

A guide to the composition and functions of the extracellular matrix.

Extracellular matrix (ECM) is a dynamic 3-dimensional network of macromolecules that provides structural support for the cells and tissues. Accumulated knowledge clearly demonstrated over the last decade that ECM plays key regulatory roles since it orchestrates cell signaling, functions, properties and morphology. Extracellularly secreted as well as cell-bound factors are among the major members of the ECM family. Proteins/glycoproteins, such as collagens, elastin, laminins and tenascins, proteoglycans and glycosaminoglycans, hyaluronan, and their cell receptors such as CD44 and integrins, responsible for cell adhesion, comprise a well-organized functional network with significant roles in health and disease. On the other hand, enzymes such as matrix metalloproteinases and specific glycosidases including heparanase and hyaluronidases contribute to matrix remodeling and affect human health. Several cell processes and functions, among them cell proliferation and survival, migration, differentiation, autophagy, angiogenesis, and immunity regulation are affected by certain matrix components. Structural alterations have been also well associated with disease progression. This guide on the composition and functions of the ECM gives a broad overview of the matrisome, the major ECM macromolecules, and their interaction networks within the ECM and with the cell surface, summarizes their main structural features and their roles in tissue organization and cell functions, and emphasizes the importance of specific ECM constituents in disease development and progression as well as the advances in molecular targeting of ECM to design new therapeutic strategies.

Brown spider venom toxins: what are the functions of astacins, serine proteases, hyaluronidases, allergens, TCTP, serpins and knottins?

Accidents caused by the bites of brown spiders (Loxosceles) generate a clinical condition that often includes a threatening necrotic skin lesion near the bite site along with a remarkable inflammatory response. Systemic disorders such as hemolysis, thrombocytopenia, and acute renal failure may occur, but are much less frequent than the local damage. It is already known that phospholipases D, highly expressed toxins in Loxosceles venom, can induce most of these injuries. However, this spider venom has a great range of toxins that probably act synergistically to enhance toxicity. The other protein classes remain poorly explored due to the difficulty in obtaining sufficient amounts of them for a thorough investigation. They include astacins (metalloproteases), serine proteases, knottins, translationally controlled tumor proteins (TCTP), hyaluronidases, allergens and serpins. It has already been shown that some of them, according to their characteristics, may participate to some extent in the development of loxoscelism. In addition, all of these toxins present potential application in several areas. The present review article summarizes information regarding some functional aspects of the protein classes listed above, discusses the directions that could be taken to materialize a comprehensive investigation on each of these toxins as well as highlights the importance of exploring the full venom repertoire.(AU)

[Comparative characteristics of enzymatic hydrolysis methods for isolating toxic substances from blood and hair]. / Sravnitel'naya kharakteristika metodik fermentativnogo gidroliza dlya izolirovaniya toksichnykh veshchestv iz tsel'noi krovi i volos.

The aim of study was to develop methods for the hydrolysis with hyaluronidases for the isolation of drugs (phenobarbital, diphenhydramine hydrochloride and phenibut) from biological objects (blood, hair) and to compare their effectivenes with previously developed methods of enzymatic hydrolysis. The studies were carried out using model «blood - model drug substance (MDS)¼, a natural and artificially colored wool (hair) of laboratory animals - guinea pigs of white, red and black natural colors, which were daily given a solution of MDS and then were subjected to cosmetic effects - coloring. The isolation of MDS from the model complex «blood - MDS¼ and from wool was carried out using the developed methods of hydrolysis with proteolytic enzymes (papain, chymopsin and chymotrypsin) and hyaluronidase. Phenobarbital and diphenhydramine from hydrolysates were extracted by liquid-liquid extraction method, phenibut - by direct freezing extraction with acetonitrile. The analysis of the extracts was carried out by gas chromatography with mass selective detection. The results showed that the developed methods of enzymatic hydrolysis can be recommended for isolating substances of various properties (acidic, amphoteric and alkaline) from blood, natural and artificially colored wool (hair).

Hyaluronan in the Tumor Microenvironment.

The extracellular matrix is part of the microenvironment and its functions are associated with the physical and chemical properties of the tissue. Among the extracellular components, the glycosaminoglycan hyaluronan is a key component, defining both the physical and biochemical characteristics of the healthy matrices. The hyaluronan metabolism is strictly regulated in physiological conditions, but in the tumoral tissues, its expression, size and binding proteins interaction are dysregulated. Hyaluronan from the tumor microenvironment promotes tumor cell proliferation, invasion, immune evasion, stemness alterations as well as drug resistance. This chapter describes data regarding novel concepts of hyaluronan functions in the tumor. Additionally, we discuss potential clinical applications of targeting HA metabolism in cancer therapy.

Snake Venom Hemotoxic Enzymes: Biochemical Comparison between Crotalus Species from Central Mexico.

Snakebite envenoming is a serious medical problem in different areas of the world. In Latin America, the major prevalence is due to snakes of the family Viperidae, where rattlesnakes (Crotalus) are included. They produce hemotoxic venom which causes bleeding, tissue degradation and necrosis. Each venom has several enzymatic activities, producing different effects in the envenoming, doing its clinical effects difficult to study. Comparison between venom molecules is also difficult when different techniques are used, and therefore, their identification/characterization using the same methodology is necessary. In this work, a general biochemical characterization in snake venom of serine proteases (SVSP), phospholipases A2 (PLA2), metalloproteases (SVMP) and hyaluronidases (SVH) of Crotalus aquilus (Ca), Crotalus polystictus (Cp) and Crotalus molossus nigrescens (Cmn) was done. Differences in protein pattern, enzyme content and enzymatic activities were observed. All the venoms showed high PLA2 activity, high molecular weight SVSP, and a wide variety of SVMP and SVH forms. Ca and Cp showed the highest enzymatic activities of SVMP and SVSP trypsin-like and chymotrypsin-like, whereas Cmn showed the highest SVH and similar PLA2 activity with Ca. All the venoms showed peptides with similar molecular weight to crotamine-like myotoxins. No previous biochemical characterization of C. aquilus has been reported and there are no previous analyses that include these four protein families in these Crotalus venoms.

The Complex Interplay Between Extracellular Matrix and Cells in Tissues.

Extracellular matrix (ECM) maintains the structural integrity of tissues and regulates cell and tissue functions. ECM is comprised of fibrillar proteins, proteoglycans (PGs), glycosaminoglycans, and glycoproteins, creating a heterogeneous but well-orchestrated network. This network communicates with resident cells via cell-surface receptors. In particular, integrins, CD44, discoidin domain receptors, and cell-surface PGs and additionally voltage-gated ion channels can interact with ECM components, regulating signaling cascades as well as cytoskeleton configuration. The interplay of ECM with recipient cells is enriched by the extracellular vesicles, as they accommodate ECM, signaling, and cytoskeleton molecules in their cargo. Along with the numerous biological properties that ECM can modify, autophagy and angiogenesis, which are critical for tissue homeostasis, are included. Throughout development and disease onset and progression, ECM endures rearrangement to fulfill cellular requirements. The main responsible molecules for tissue remodeling are ECM-degrading enzymes including matrix metalloproteinases, plasminogen activators, cathepsins, and hyaluronidases, which can modify the ECM structure and function in a dynamic mode. A brief summary of the complex interplay between ECM macromolecules and cells in tissues and the contribution of ECM in tissue homeostasis and diseases is given.

Hyaluronic Acid in the Third Millennium.

Since its first isolation in 1934, hyaluronic acid (HA) has been studied across a variety of research areas. This unbranched glycosaminoglycan consisting of repeating disaccharide units of N-acetyl-d-glucosamine and d-glucuronic acid is almost ubiquitous in humans and in other vertebrates. HA is involved in many key processes, including cell signaling, wound reparation, tissue regeneration, morphogenesis, matrix organization and pathobiology, and has unique physico-chemical properties, such as biocompatibility, biodegradability, mucoadhesivity, hygroscopicity and viscoelasticity. For these reasons, exogenous HA has been investigated as a drug delivery system and treatment in cancer, ophthalmology, arthrology, pneumology, rhinology, urology, aesthetic medicine and cosmetics. To improve and customize its properties and applications, HA can be subjected to chemical modifications: conjugation and crosslinking. The present review gives an overview regarding HA, describing its history, physico-chemical, structural and hydrodynamic properties and biology (occurrence, biosynthesis (by hyaluronan synthases), degradation (by hyaluronidases and oxidative stress), roles, mechanisms of action and receptors). Furthermore, both conventional and recently emerging methods developed for the industrial production of HA and its chemical derivatization are presented. Finally, the medical, pharmaceutical and cosmetic applications of HA and its derivatives are reviewed, reporting examples of HA-based products that currently are on the market or are undergoing further investigations.

Isolation and characterization of Conohyal-P1, a hyaluronidase from the injected venom of Conus purpurascens.

Hyaluronidases are ubiquitous enzymes commonly found in venom and their main function is to degrade hyaluran, which is the major glycosaminoglycan of the extracellular matrix in animal tissues. Here we describe the purification and characterization of a 60kDa hyaluronidase found in the injected venom from Conus purpurascens, Conohyal-P1. Using a combined strategy based on transcriptomic and proteomic analysis, we determined the Conohyal-P1 sequence. Conohyal-P1 has conserved consensus catalytic and positioning domain residues characteristic of hyaluronidases and a C-terminus EGF-like domain. Additionally, the enzyme is expressed as a mixture of glycosylated isoforms at five asparagine sites. The activity of the native Conohyal-P1 was assess MS-based methods and confirmed by classical turbidimetric methods. The MS-based assay is particularly sensitive and provides the first detailed analysis of a venom hyaluronidase activity monitored with this method. The discovery of new hyaluronidases and the development of techniques to evaluate their performance can advance several therapeutic procedures, as these enzymes are widely used for enhanced drug delivery applications. BIOLOGICAL SIGNIFICANCE: Cone snail venom is a remarkable source of therapeutically important molecules, as is the case of conotoxins, which have undergone extensive clinical trials for several applications. In addition to the conotoxins, a large array of proteins have been reported in the venom of several species of cone snails, including enzymes that were found in dissected and injected Conus venom. Here we describe the isolation and characterization of the hyaluronidase Conohyal-P1 from the injected venom of C. purpurascens. We employed a combined transcriptomic and proteomic analysis to obtain the full sequence of this hyaluronidase. The activity of Conohyal-P1 was assessed by a mass spectrometry-based method, which provide the first detailed venom hyaluronidase activity analysis monitored by mass spectrometry allowing the visualization of the substrate degradation by the enzyme.

Albizia lebbeck seed methanolic extract as a complementary therapy to manage local toxicity of Echis carinatus venom in a murine model.

CONTEXT AND OBJECTIVE: Viperid venom-induced chronic local-toxicity continues even after anti-snake venom treatment. Therefore, traditional antidote Albizia lebbeck L. (Fabaceae) seed extract was tested against Echis carinatus S. (Viperidae) venom (ECV)-induced local toxicity to evaluate its complementary remedy. MATERIALS AND METHODS: Soxhlet extraction of A. lebbeck seeds was performed with the increasing polarity of solvents (n-hexane to water); the extract was screened for phytochemicals (alkaloids, anthraquinones, flavonoids, glycosides, phenolics, saponins, steroids and tannins). In preliminary in vitro analysis, A. lebbeck methanolic extract (ALME) demonstrated significant inhibition of ECV proteases, the major enzyme-toxin responsible for local- toxicity. Therefore, in vitro neutralizing potential of ALME was further evaluated against hyaluronidases and phospholipase A2 (1:1-1:100 w/w). In addition, alleviation of ECV induced characteristic local- toxicity [haemorrhage (i.d.) and myotoxicity (i.m.)] was determined in mice. RESULTS: ALME contained high concentrations of phenolics and flavonoids and demonstrated significant in vitro inhibition of ECV protease (IC50 = 36.32 µg, p < 0.0001) and hyaluronidase (IC50 = 91.95 µg, p < 0.0001) at 1:100 w/w. ALME significantly neutralized ECV induced haemorrhage (ED50 = 26.37 µg, p < 0.0001) and myotoxicity by significantly reducing serum creatinine kinase (ED50 = 37.5 µg, p < 0.0001) and lactate dehydrogenase (ED50 = 31.44 µg, p = 0.0021) levels at 1:50 w/w. DISCUSSION AND CONCLUSION: ALME demonstrated significant neutralization of ECV enzymes that contribute in local tissue damage and haemostatic alterations. The study scientifically supports the anecdotal use of A. lebbeck in complementary medicine and identifies ALME as principle fraction responsible for antivenom properties.